The present invention relates generally to methods for identifying microorganisms present in fruit juices generally and more specifically in wine. More specifically, the invention relates to methods of isolating nucleic acids present in fruit juice in order that they may be successfully amplified and/or detected by molecular amplification and detection methods including that known to the art such as polymerase chain reaction (PCR).
The fermentation of grape juice (must) to produce wine is a complex biological process in which yeasts, most typically Saccharomyces cerevisiae, are used to convert the sugar content of grape must to ethanol, water, and carbon dioxide. Not only do the many different strains of Saccharomyces cerevisiae differ with respect to their metabolic processes and products, but grape must typically comprises a multiplicity of other microorganisms including other fungi, yeasts, and bacteria which react with the grape must during fermentation to produce various metabolic products including aromatic and flavoring compounds. Such products can influence the sensory character of the resulting fermentation product in both positive and negative ways. Conversely, the presence of unwanted microorganisms can lead to undesirable flavors or spoilage. In some cases contaminated must can produce fermentation products characterized by undesirable commercial attributes, affecting flavor, color, and/or mouthfeel. It is thus of intense interest to the vintner to know whether the fermentation is characterized by such microbial contaminants. Similarly, the presence of contaminating microorganisms in the finished wine product is of concern to the wine producer and there exists a desire to ensure the absence of such contaminants. It is also crucial to know if microbes are present after the fermentation has been completed, as the majority of spoilage occurs during the aging and storage of the finished product.
Alternatively, where it is desired to inoculate grape must with a malolactic fermentation bacterium to carry out malolactic fermentation, it is useful to evaluate the growth of the inoculated strain and determine whether the fermentation medium need be reinoculated. Previously, to determine the presence and identity of microorganisms in the grape must, it has been necessary to culture samples of the grape must to xe2x80x9cgrow upxe2x80x9d cell cultures in different selective media. The presence of microbial contaminants can then be identified morphologically or otherwise. Not only is morphological identification sometimes uncertain, but in the case of Dekkera sp., several days are required to culture a sample to determine its presence.
More recently, advances in molecular biology have made it possible to identify microorganisms by amplifying and/or detecting nucleic acid sequences which are uniquely characteristic of those microorganisms by methods such as polymerase chain reaction (PCR), strand displacement amplification (SDA), ligase chain reaction (LCR), RFLPs, direct sequencing (dideoxy method), direct hybridization, and immuno-based assay techniques. In this regard, much recent work has been conducted in the identification of polynucleic acid sequences which are uniquely characteristic of microorganisms present in wine fermentations. For example, ribosomal DNA (rDNA) genes are particularly useful as targets for molecular probes and PCR primers because of their high copy number. Moreover, non-transcribed and transcribed spacer sequences associated with ribosomal genes are usually poorly conserved and thus are useful for the detection and identification of different closely related fungal pathogens. The internal transcribed spacer (ITS) region lies between the 18S and 28S rRNA genes and contains two variable non-coding spacers referred to as ITS1 and ITS2 separated by the 5.85 gene. In addition, the transcriptional units are separated by non-transcribed spacer (NTS) sequences. The ITS and NTS sequences are particularly suitable for the detection of fungal pathogens. PCT International Application WO 99/46405 discloses unique DNA sequences which are useful in the identification of microorganisms involved in fermentations such as Saccharomyces cerevisiae, Saccharomycodes ludwigii, Dekkera sp., Botrytis cinerea, Penicillium sp., Hanseniaspora guilliermondii, Debaryomyces carsonii, Pichia anomala, Pichia kluyveri, and Candida krusei. See also Bartowsky, et al., Australian J. of Grape and Wine Research 5:39-44 (1999) which describes the use of PCR for specific detection of the malolactic fermentation bacterium Oenococcus oeni (Leuconostoc oenos) isolated from bacterial culture.
Despite advances in the molecular identification of microorganisms associated with wine, fruit juice, and fruit-juice fermentations, it has not been possible to successfully conduct a nucleic acid amplification/detection method such as PCR amplification directly on a wine sample. While all the reasons for this remain unclear, they may include concentration effects and may also be related to the presence of potentially inhibitory compounds in the wine, among other factors. It thus remains the case that microorganisms present in wine samples must be subjected to a culturing step in order to provide sufficient quantities of cells to serve as a source of nucleic acids for amplification by PCR or other molecular amplification or detection techniques. The requirement that sample cells be amplified by cell-culturing constitutes a significant limitation to the utility of molecular identification methods because of the time delay (typically several days) inherent in cell-culturing. The vintner is therefore unable to apply real-time adjustments to the fermentation to take advantage of the information provided by the molecular identification techniques. In the case of the presence of undesired microorganisms in a wine fermentation, the loss of time in which to address the outbreak may mean the difference between successfully applying remediation techniques to salvage the fermentation and degradation or ruination of the fermentation batch. Real-time identification of potential microbial problems will enable the proactive, rather than reactive, use of one or more targeted treatments such as cooling, filtering, application of clarification and/or fining agents, anti-microbial substances, and reinoculation, thereby increasing their effectiveness. For this reason, there remains a desire in the art for methods for conducting xe2x80x9creal-timexe2x80x9d PCR or other amplification techniques not requiring an intermediate cell-culturing step to multiply the cell sample quantity.
While the preceding discussion has focused on the need for improved nucleic acid amplification and/or detection methods for application to the wine industry, it is apparent that there exist similar needs for testing of other fruit juices. Such a need is important in the cider industry but is particularly acute given the growth in popularity of xe2x80x9corganicxe2x80x9d and other xe2x80x9cnaturalxe2x80x9d fruit juices and ciders which are sometimes not subject to pasteurization or treated with preservatives.
Attempts have been made to use conventional cellulosic filters to isolate and concentrate organisms within grape must or wine in order to perform PCR or other amplification or nucleic acid detection techniques on the retentate. Such methods have not been successful and the failure of those methods may relate to the retention of inhibitory compounds within the retentate. More recently, methods such as those of DiMichele and Lewis, ASBC Journal, 51:2, 63-66 (1993) have shown promise in conducting real-time PCR for the identification of organisms involved in the fermentation of beer. According to DiMichele and Lewis, Lactobacillus brevis, L. casei, and L. plantarum were isolated from water and beer by membrane filtration and membrane dissolution using a dissolvable polycarbonate membrane having a pore size of 0.45 micron. Regions of the 16S rRNA sequences unique to each organism were used to generate species-specific PCR products, which were then visualized by gel electrophoresis. The DiMichele reference noted that beer contained a PCR inhibitor that decreased sensitivity.
Despite the promising results reported in beer, the methods of DiMichele et al. would not be expected to work successfully in fruit juices and wine because of differences in the chemical compositions of fruit juices compared with beer. Such differences include the presence of high concentrations of different compounds which can act to interfere with the nucleic acid hybridization and/or chain extension steps required by nucleic acid detection and amplification methods. In particular, it is believed that the acidic pH and high sugar content characteristic of fruit juices may act to interfere with nucleic acid hybridization. Further, the presence of high levels of sediment and particularly the presence of high levels of polyphenolic compounds (e.g. tannins) in juices such as grape juice is suspected to interfere with nucleic acid hybridization and chain extension. Accordingly, there remains a desire in the art for methods by which rapid xe2x80x9creal-timexe2x80x9d molecular amplification and/or detection methods can be practiced in fruit juices including grape juice and wine samples.
The present invention relates to the discovery that PCR and other nucleic acid amplification and detection methods can effectively be practiced on nucleic acids derived from the cells of microorganisms isolated from fruit juices generally and particularly from grape juice, wine must, and wine (collectively, referred to herein as xe2x80x9cwinexe2x80x9d) without the necessity of culturing those microorganisms. All varieties of wine may be successfully analyzed according to the method of the invention including white, pink, and red varieties. Specifically, the invention provides a method of isolating cells of target microorganisms present in fruit juice in a form suitable for amplification of the nucleic acids contained therein comprising the steps of: (a) passing fruit juice through a dissolvable filter having a pore size selected to retain said target microorganisms; (b) extracting the retentate from the filter with an organic solvent selected to dissolve said filter while not interfering with the integrity of nucleic acids present in the cells of said test microorganisms; (c) subjecting the extracted cells to an organic wash step; (d) subjecting the extracted cells to an aqueous wash step; and (e) concentrating said cells. In addition, the invention provides alternative methods of (f) isolating nucleic acids from said concentrated cells or (f) subjecting nucleic acids present in the concentrated cells to an amplification and/or detection procedure including those selected from the group consisting of polymerase chain reaction, strand displacement amplification, ligase chain reaction, restriction fragment length polymorphism, dideoxy sequencing, immuno-based sequencing, and hybridization. In some cases, an amplification procedure can be followed by g) detecting the presence of said target nucleic acid sequence. The detection of such target nucleic acids can be carried out by methods well known to the art such as by the use of nucleic acid probes capable of specifically hybridizing with the target nucleic acid sequence. Such probes may be used according to methods well known to the art including, but not limited to, xe2x80x9cdip sticksxe2x80x9d and other solid phase detection assay devices. The method of the invention is applicable to fruit juices of all sorts but is particularly preferred for use wherein the fruit juice is selected from the group consisting of grape, apple, cranberry, plum, tomato, kiwis, strawberry, raspberry, and citrus juices including lemon, orange, and grapefruit juices. Most preferably the fruit juice is grape juice and is fermented grape juice such as wine or wine must.
The invention also provides kits for isolating cells of microorganisms present in fruit juice (including wine and wine must) in a form suitable for amplification of the nucleic acids contained therein comprising: (a) a dissolvable filter having a pore size of from 0.4 micron to 1 micron with pore sizes of from 0.6 micron to 1 micron being preferred; and (b) an organic solvent selected to dissolve said filter while not interfering with the integrity of nucleic acids present in cells of the target microorganisms. Other kits according to the invention include a kit for amplifying nucleic acids from microorganisms present in fruit juice comprising: (a) a dissolvable filter having a pore size of from 0.4 micron to 1 micron being preferred with pore sizes of from 0.6 micron to 1 micron being preferred; and (b) an oligonucleotide primer selected to specifically hybridize with a polynucleic acid characteristic of a microorganism present in fruit juice. Preferred kits according to the invention further provide: c) a nucleic acid probe capable of hybridizing to the target nucleic acid. Preferred kits according to the invention further include a polyphenolic binding agent which is preferably selected from the group consisting of polyvinypolypyrrolidone and polyvinylpyrrolidone.
The present invention provides methods for isolating cells of microorganisms present in fruit juice in a form suitable for amplification and/or detection of nucleic acids contained therein. In this manner, microorganisms present in the fruit juices can be identified by the detection (optionally preceded by amplification) of nucleic acid sequences specific to given microorganisms. As used herein, the term xe2x80x9cjuicexe2x80x9d refers to fermented and unfermented grape and other fruit juices and concentrates and extracts, and is inclusive of those juices and extracts at any stage from and including fresh fruit to a finished fermented wine product. The methods of the present invention have proven useful in isolating microorganisms and amplifying DNA from cells in grapefruit, lemon, and apple juices but are particularly useful when applied to grape must during the fermentation process but are not limited thereto and are also directed to analysis of finished wines.
The methods of the invention involve the step of passing fruit juice through a dissolvable filter characterized by a defined pore size. Such dissolvable (or soluble) filters are dissolvable in organic solvents but retain their structural integrity in aqueous fluids including fruit juices and wine. Polycarbonate filters such as those manufactured by Whatman (Nuclepore(copyright)PC) are particularly useful in practice of the invention as they can be dissolved in organic solvents such as chloroform, methylene chloride, and N-methyl-2-pyrrolidone which do not interfere with the integrity of nucleic acids present in the cells of target organisms. By not xe2x80x9cinterfere with the integrity of nucleic acids,xe2x80x9d it is meant that an organic solvent which does not alter the structure of polynucleic acids such that they are cleaved or otherwise modified or altered in a way that interferes with nucleic acid hybridization, chain extension, recognition of original polyacid identity or other molecular techniques used to amplify or detect polynucleic acid sequences.
Other dissolvable filters that can be used according to the invention include mixed cellulose esters (MCE) such as available from Whatman as Membra-Fil(copyright) (MF) that are soluble in organic solvents such as acetone, dimethylformamide, dimethylacetamide, N-methyl-2-pyrrolidone, and polyester (PE) filters such as Nuclepore (copyright) (PE) that are soluble in organic solvents which do not interfere with the integrity of nucleic acids such as hexafluoroisopropanol, M-cresol, O-chlorophenol, and phenol/tetrachloroethane (1:1). The dissolvable filters are characterized by a pore size adapted to retain fruit juice and wine-related target microorganisms in order that their DNA may be extracted, amplified, and analyzed. Preferred nominal pore sizes include those greater than 0.4 micron with preferred filters having a pore size from about 0.6 to 1 micron and filters having a nominal pore size of about 0.8 micron being particularly useful in wine analysis because they are capable of retaining live yeasts and other fungi but not bacteria.
Upon filtering of the fruit juice sample through the dissolvable filter and discarding of the filtrate, the retentate present on and within the dissolvable filter material is extracted with an organic solvent selected to dissolve the dissolvable filter while not interfering with the integrity of nucleic acids. While not necessary for practice of the invention, it is also preferred that the organic solvent not unduly damage or lyse the cells of microorganisms retained on the filter. Suitable solvents for use in practice of the invention include N-methyl-2-pyrrolidone, methylene chloride, or chloroform. Further, the organic solvent should be selected such that it does not degrade the DNA contained within the microorganisms in a manner such that it cannot be amplified. While the identity of suitable solvents will be determined by the character of the filter, a preferred solvent for use with polycarbonate filters is a blend comprising phenol, chloroform, and isoamyl alcohol at a ratio of 25:24:1. The extraction is preferably carried out at room temperature and at a pH of about 7.8 because (DNA only, not RNA) nucleic acids will partition into the organic phase at acidic pH (i.e., at acid pH, DNA goes into organic phase, but RNA stays in aqueous). Those of skill in the art will recognize that other blends of these components and of other organic components will serve to dissolve the filter and suitably extract the retained microorganisms in a manner which does not affect the ability to extract and amplify nucleic acid sequences in the microorganisms.
The product of the extraction step comprising the organic solvent, an aqueous solvent (water), dissolved filter, sediment, and extracted microorganisms is then vortexed and centrifuged briefly to separate the aqueous and organic phases. The majority of the extracted cells are present in the aqueous phase which is then transferred to a fresh tube and is subjected to a number of washing steps including one or more wash steps with an organic solvent and one or more wash steps with aqueous and organic solvents to produce a concentrated cell pellet. The organic wash step is preferably carried out with an organic solvent such as phenol or chloroform or mixtures of organic solvents such as a phenol:chloroform:isoamyl alcohol (25:24:1) solution. The aqueous wash step is preferably carried out with deionized water but those of skill in the art will recognize that other aqueous solvents may be used to wash and resuspend the isolated cells and nucleic acids. This cell pellet may then be resuspended according to methods known in the art and the nucleic acids present in the cells used as templates for amplification techniques.
According to one preferred method of the invention, 250 mL of wine is filtered through a 47-mm polycarbonate filter membrane characterized by a pore size of 0.8 micron. The filter containing microorganisms present in the wine is dissolved in a solvent comprising 0.7 mL of deionized water and 1.05 mL of an organic solvent comprising phenol:chloroform:isoamyl alcohol at a ratio of 25:24:1. The product of this extraction comprising sediment, microorganisms, dissolved filter, and solvents is then vortexed and centrifuged briefly to separate aqueous and organic phases. The aqueous phase of the extract is then transferred to a fresh tube and washed with an equal amount of phenol. The product is then mixed and centrifuged briefly to separate organic from aqueous layers. The aqueous phase is then transferred to a fresh tube and washed with an equal amount of phenol:chloroform:isoamyl alcohol (25:24:1). The mixture is then centrifuged briefly to separate layers. The aqueous phase is transferred to a fresh tube and washed with an equal amount of chloroform. The mixture is then centrifuged briefly to separate layers. The aqueous layer is then extracted, and 2.8 mL of deionized water is added and mixed by inversion. The mixture is then centrifuged for five minutes at 5000xc3x97g. The liquid is then discharged down to 100 xcexcL without disturbing the pellet and the pellet is gently resuspended in 2.8 mL of deionized water and the centrifugation step is repeated. The resulting pellet is then resuspended and used as a template in a PCR procedure using 0.4% polyvinylpolypyrrolidone.
The following examples are presented to more clearly illustrate the invention. Examples 1 through 16 are directed to use of conventional filtration methods to isolate microorganism (yeast) cells from wine in preparation for practice of a polymerase chain reaction (PCR) amplification of yeast DNA sequences. Practice of these conventional filtration techniques proved unable to provide sufficient yeast DNA in condition for amplification by PCR despite modification of the basic filtration method of Example 1 by use of surfactants (Examples 2 and 4); adjustment of wine pH (Examples 3 and 4) and use of sonication and shaking methods (Examples 5 through 16). Examples 17 through 19 are directed to use of the method described by DiMichele and Lewis, ASBC Journal, 51(2):63-66 (1993) for conducting PCR of yeast DNA in the wine using a dissolvable polycarbonate filter instead of bacteria DNA in beer as taught by DiMchele and Lewis. While there was evidence of weak sporadic but unreproducible amplification when the method was applied to a white wine (Example 17), there was no sign of amplification when applied to pink (Example 18) or red (Example 19) wines. The methods of Examples 17 through 19 differed from that described by DiMichele and Lewis in that the pore size of the polycarbonate filter was 0.8 micron instead of 0.45 micron. Examples 20 and 21 disclose methods according to the present invention including the steps of subjecting the cells extracted from the dissolvable filter to an organic wash step and to an aqueous wash step. The results of these experiments consistently generated weak but reproducible amplification signals for the yeast DNA signals for both the white (Example 20) and red (Example 21) wines. Examples 22 and 23 are directed to a preferred aspect of the invention wherein strong amplification signals were produced for red wines when a polyphenolic binding agent (polyvinylpolypyrrolidone) was incorporated into the PCR buffer.